Multidrug resistance (MDR) efflux pumps and biofilm formation are two mechanisms by which bacteria can evade the action of many antimicrobials. MDR efflux pumps confer low level multidrug resistance and are over-expressed in MDR clinical isolates. Biofilms are three dimensional, complex communities of bacteria encased in a self produced extra cellular matrix. Biofilms protect the bacteria within them both physically by acting as a barrier to any external threats, and metabolically by containing high proportions of persister cells. This thesis explores the link between MDR efflux and biofilm formation in S. Typhimurium and shows that genetic inactivation of any one of the nine MDR efflux systems results in a biofilm defect. We found that the transcriptional repression of curli, an essential component of the Salmonella matrix, is the reason for the efflux mutants' inability to form a biofilm. The biofilm defect in a tolC mutant was rescued by inactivation of a gene encoding an osmolarity sensor, envZ, suggesting that membrane stress response is a possible link between efflux and biofilm formation. Chemical efflux inhibitors (EIs) were found to impart curli repression and cause subsequent biofilm defect. This finding is clinically important as biofilms are a major cause of infection EIs could be a potential novel anti-biofilm therapy.

Recent evidence indicates that immune cells within adipose tissues can drive the formation of ectopic lymphoid structures, known as Fat Associated Lymphoid Clusters (FALC). FALC support B-cell antibody production in response to infection and inflammation. This investigation explores the immune cell composition and role of different adipose tissues both in steady state and during immune responses in mice. Firstly, a detailed analysis of the immune cell composition of peritoneal adipose tissues was performed. To investigate the function and migratory properties of these tissue-resident cells, cytokine and chemokine receptor expression was then assessed. How immune cells in adipose tissues responded to infection was examined using an intestinal helminth infection with the parasite Heligmosomoides polygyrus. Adipose tissues predominantly contained regulatory T cells, invariant natural killer T cells and group 2 Innate Lymphoid Cells (ILC2s). Significant differences were observed in composition, cell surface markers and cytokine production of ILC2s between adipose depots and secondary lymphoid tissues, indicating that tissue specific signals can direct ILC2 responses. Finally, increases were observed in ILC2 and FALC numbers in the mesenteries of WT mice following parasite infection. These data indicate that immune cells within adipose tissues respond to infection and may contribute to immune responses.

The first project presented here was generated around 1 calcitriol (1,25D₃), and novel low-calcemic analogues (VDAs). The hypothesis of this study was that VDAs may be effective in Diffuse Large B cell Lymphoma. It has been demonstrated that 1,25D₃ and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. Additionally, 1,25D₃ and VDAs used in a combination with antidepressant clomipramine displayed concentration anti-stimulatory actions upon activated normal B-cells. The second part of the study is focused around a novel immunomodulator CX1001, data from which is currently confidential. The third project has been built around GPR61. I have demonstrated that myc-tagged GPR61 is expressed in the cell membrane and is subject to N-glycosylation. The N12S mutant of GPR61, which is not subject to N-glycosylation, is also expressed on the cell surface. Treating the cells with tunicamycin reduces cell membrane expression of non-mutated and mutated proteins. Furthermore, GPR61 mRNA and protein are expressed in white blood cells with significantly increased level of expression of the protein in Th17 cells.

G-protein-coupled receptors (GPCRs) are major pharmaceutical drug targets due to their crucial role in cell signalling. The neurohypophysial peptide hormones [Arg⁸] vasopressin (AVP) and oxytocin (OT) signal through a subfamily of GPCRs, comprising the AVP receptors (V₁a R, V₁bR and V₂R) and the OT receptor (OTR). The aim of this work was to understand the molecular basis of receptor activation by defining exact contacts between agonist and receptor. Molecular modelling suggested that two conserved negatively-charged residues may be important for agonist binding and receptor activation. Interactions between agonists and the human AVP/OT receptors were probed using a combination of site-directed mutagenesis of the receptors and ligands incorporating modifications at specific points. The wild-type (WT) and mutant receptors were expressed in HEK 293T cells and pharmacologically characterised with respect to ligand binding, receptor activation, cell surface expression and ligand-induced internalisation when stimulated by endogenous, or modified, ligands. Mutual exchange of functional groups revealed direct interactions between AVP/OT/[Arg⁸] vasotocin (AVT; a chimera of AVP and OT) and the human AVP/OT receptors required for receptor activation. These studies advance current understanding of the molecular basis of receptor activation and have the potential to facilitate future rational drug design.

This thesis consists of two volumes: a clinical and a research volume. The clinical volume contains five clinical practice reports; the research volume contains a systematic review and an empirical study. The systematic review explores the effectiveness of cognitive rehabilitation of problem-solving, planning and reasoning in people with traumatic brain injury. The findings suggest that Short-Term Executive Plus (STEP) can decrease executive dysfunction and improve problem-solving, however, more research would strengthen the findings. Evidence for the use of ‘gist reasoning’ training is growing. Training multitasking remains an approach with insufficient evidence to support it. Finally, although the findings in relation to telephone counselling seem promising, more research could help to clarify its effectiveness. The empirical study aimed to establish whether the Iowa Gambling Task (IGT) adds to the predictive potential of three tests of executive function in the assessment of capacity to take part in research in people with acquired brain injury. Stroop errors on the inhibition task and Wisconsin Card Sorting Test (WCST) total errors emerged as potential screening tools; the IGT was not among the predictors. Suggestions for future research are provided. Replication with a larger sample is needed to confirm the findings.

Graphene oxide (GO) and reduced GO (r-GO) nanomaterials exhibit great potential for several biomedical applications. Of foremost importance is to determine any potential health hazards related in their exposure. In this research, we hypothesised that the different material properties evidenced by GO and r-GO would elicit different biological responses. The first objective of this work was to synthesize Go and r-GO and characterize their physiochemical properties. The second aim was to investigate whether the two-distinct surface chemistries of GO and r-GO influenced their biological effect. The potential toxicity of these nanomaterials was investigated using the normal lung fibroplast cell line MRC-5 and cancerous epithelial lung cell line A549. The cytotoxicity of graphene derivatives was concentration-, time- and cell-dependent and varied according to the material used. Thus, the surface chemistry of graphene plays a critical role in its biocompatibility. Non-cancerous cells had a higher sensitivity to GO cytotoxicity than cancer cells. R-GO was highly biocompatible to MRC-5 cells and for A549 cells had a minimal effect of cell viability. At 37C˚, GO and r-GO were moderately hemolyric at concentration of 125 µg/ml and highly haemolytic at concentration of 300 µg/ml. Exposure of cells to both graphene derivatives led to reactive oxygen species (RO5) generation without genotoxicity. GO, but not r-GO, led to autophagy in both cell lines, possibly inhibiting the PIP3-Akt/mTOR pathway. For both cell lines and at non-lethal concentrations, GO downregulated the expression of glycogen synthase kinase-3 (GSK-3ß). GO was also found to dysregulate both Wnt/b-catenin and Akt cell signalling pathways which are vital for cellular function. The finding relating to cell signalling provide an insight to the safety of GO which is important to its use in cancer therapy.

Patients termed to have radioiodine-refractory differentiated thyroid cancer (RR-DTC) cannot accumulate sufficient radioiodine for a therapeutic response due to sodium iodide symporter (NIS) dysregulation via diminished expression and/or altered plasma membrane localisation. Previous studies identified novel therapeutics to increase NIS expression, but these are associated with poor tolerance and resistance. Meanwhile, regulation of NIS plasma membrane localisation remains poorly defined and, despite protein-protein interactions being well-described to modulate trafficking events, the NIS interactome is limited. Here, two novel functional NIS interactors – ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP) – were identified by mass spectrometry, which positively and negatively modulated radioiodine uptake respectively. In papillary thyroid cancer (PTC), ARF4 is downregulated, and VCP overexpressed, which may provide a putative explanation for repressed NIS function. Subsequent investigations identified co-localisation between ARF4 and NIS at the Golgi as well as co-incident trafficking at the plasma membrane, which led to the hypothesis that ARF4 promotes NIS trafficking to the plasma membrane. In contrast, VCP decreased NIS protein expression, which was suggestive of a role for VCP in NIS processing and degradation. Pharmacological inhibitors Eeyarestatin-1 and NMS-873 overcame VCP inhibition of NIS function, which may highlight a novel therapeutic strategy for RR-DTC.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, leading to hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. A vaccine is currently not available, while the standard of care is effective in only 50% of treated patients. The first specific anti-HCV drugs have been recently approved, and new classes of targeted agents are under clinical trials/investigation. Nevertheless, improved treatment strategies are needed, in order to bypass the rapid emergence of resistance. All the viral non-structural proteins are a possible target for the identification of novel and selective antivirals. Among them, the NS3 helicase is still underexploited, with no known inhibitor under pre-clinical or clinical development. This enzyme plays a crucial role in the virus life cycle: it catalyses the separation of double-stranded RNA strands, which is necessary for genome amplification and translation. Due to its essential function, the NS3 helicase was chosen as a target for the identification of new, specific anti-HCV compounds. Different computer-aided techniques were employed to identify potential smallmolecule inhibitors of the enzyme. Two structure-based virtual screenings of commercially available compounds were performed on the main nucleic acid binding site. A series of candidate inhibitors was evaluated in the HCV replicon assay, yielding two primary hits with low μM activity. Secondly, the model of the one known inhibitor co-crystallised with the enzyme was used as a starting point for a shape-comparison screening of small molecule libraries. A new series of compounds was selected and evaluated for anti-HCV activity, and one of them was found to inhibit the viral replication at a low μM concentration. Several new derivatives of the initial hits were synthesised, belonging to four main structural families: bis-aromatic piperazine derivatives, symmetrical phenylendiamine compounds, differently substituted thieno-pyrimidines, and triphenyl-pyrrolone analogues. Inhibition of HCV replication in the replicon assay was evaluated for the new compounds prepared and several structures showed a range of activity from low-μM to nM.

This thesis, awarded by published work represents a collection of clinical studies that advance the knowledge of why to, and how best to, administer propofol safely using an innovative technique, 'Patient Maintained Sedation' (PMS).

Background Many randomised controlled trials (RCTs) are available to support using different pharmacotherapy agents in the management of various neuropathic pain conditions. However, choosing these pharmacotherapy agents for neuropathic pain is challenging, due to the limited evidence-based knowledge to support the use of different pharmacotherapy agents in different neuropathic pain conditions. Aims The aim of this PhD is to evaluate the efficacy and safety of oral and topical pharmacotherapies for managing neuropathic pain by deriving placebo and active comparative efficacy and safety evidence from RCTs. Methods This research used three approaches to summarise and synthesise evidence from randomised controlled studies including: a systematic review of placebo and active control RCTs to summarise and criticise the current evidence in neuropathic pain; a meta-analysis and sequential analysis of eligible studies to provide a more precise estimate of the overall treatment effects; and a network meta-analysis to estimate the relative effectiveness of the most commonly used interventions in neuropathic pain. Results Systematic review Two hundred placebo and active-controlled trials met the inclusion criteria. A wide range of different treatments were studied in these trials, including anticonvulsants, antidepressants, opioids and topical capsaicin and lidocaine. Most of the included studies were parallel placebo-controlled trials and commonly lasted for 3 to 12 weeks. In addition, the vast majority of the included RCTs were conducted in participants with painful diabetic neuropathy and post-herpetic neuralgia, while only a few trials were conducted in participants with central neuropathic pain conditions. Pairwise meta-analysis Sixty seven trials were eligible for the pairwise meta analysis of efficacy outcomes. Of the anticonvulsants group pregabalin and gabapentin compared with placebo demonstrated efficacy for 50% and 30% pain reduction and global improvement in patients with neuropathic pain. The efficacy of anticonvulsants varied in different types of neuropathic pain. Gabapentin when compared against a placebo was better than a placebo in PHN and PDN, while pregabalin was significantly effective in patients with post-herpetic neuralgia (PHN) and painful diabetic neuropathy (PDN) but not in patients with HIV associated neuropathic pain. Others anticonvulsant agents, such as lamotrigine, valproic acid, topiramate, levetiracetam and oxcarbazepine, were tested in a small number of trials. These did not provide useful benefits compared with a placebo for a 50% and 30% pain reduction. Of the antidepressant group, duloxetine when compared to a placebo demonstrated efficacy for 50% and 30% pain reduction in diabetic neuropathic pain. A few active comparison trials failed to demonstrate superior efficacy of one drug over another for a 50% and 30% reduction in neuropathic pain. Trial sequential analysis To examine the reliability and conclusiveness of the available evidence, trialsequential analysis has been applied in this study. The results show convincing evidence of the efficacy of some interventions (e.g. pregabalin, gabapentin and duloxetine) to reduce pain by 50% in some neuropathic pain conditions (e.g. diabetic neuropathic pain and post-herpetic neuralgia). The continuation of RCTs of pregabalin and duloxetine in diabetic neuropathy and gabapentin in post-herpetic neuralgia is not necessary as there appears to be sufficient evidence of the efficacy of these treatments in the management diabetic neuropathic pain and post herpetic neuralgia. Further RCTs of duloxetine, pregabalin and gabapentin are however required for central neuropathic pain. In contrast, the analysis failed to provide evidence that opioids and high concentration capsaicin demonstrate a 50% pain reduction. Network meta-analysis Twenty-eight trials were eligible for the network meta-analysis. The results incorporating both direct-comparison and indirect-comparison evidence suggested that there is no superiority of duloxetine over amitriptyline, pregabalin and gabapentin in achieving at least a 30% and 50% pain reduction with a treatment duration of 7 to 12 weeks in patients with neuropathic pain conditions, such as diabetic neuropathic pain, postherpetic neuralgia and spinal cord injury. Conclusions In summary, this research has found that some good quality trials provide good evidence regarding the efficacy of duloxetine, pregabalin and gabapentin in a minority of patients with neuropathic pain. Until advancements in developing mechanism-based approaches and improved clinical trial design become available, the routine use of these medications is unlikely to be changed. This may support the hypothesis that traditional RCTs might not be a suitable method of choice to address provisional health questions in routine clinical practice.